One of the pathologic features of HIV-associated nephropathy (HIVAN) is the collapsing focal segmental glomerulosclerosis (FSGS). Glomerular visceral epithelial cells (podocytes), the key cells involved in FSGS, exhibit a disease phenotype characterized by proliferation and dedifferentiation in both human and mouse kidneys with HIVAN. HIV-1 mRNA were expressed in podocytes of HIVAN patients and of HIV-1 transgenic mice. Recently, we found that Nef, one of the HIV viral accessory proteins, is responsible for HIV-l-inuduced proliferation and loss of differentiation markers in podocytes. To understand the mechanism by which Nef induces the changes of phenotype in podocytes, we will study the different signaling pathways regulated by Nef. In the current project: 1) We will examine the down-stream signaling pathways regulated by Nef in vitro using conditionally immortalized kidney podocytes and verify the findings in primary cultures of podocytes and in vivo in podocytes of HIV-1 transgenic mice and in kidney biopsies of HIVAN patients. As suggested by our preliminary data, we will focus on Src-Stat3, Ras-cRaf-MAPK1,2, and Rac1/Cdc42-PAK pathways. 2) We will determine the role of Nef-induced signaling in generating the phenotypic changes observed in podocytes. We will determine whether dominant negative mutants for Src, Stat3, Ras, Racl, or inhibitor for MAPK1,2 prevent Nef-induced changes of phenotype in podocytes. We will also examine the co-localization of signaling molecules and proliferation or differentiation markers of podocytes in kidneys of HIV-1 transgenic mice and HIVAN patients by double staining. 3) We will investigate whether blockade of Nef expression by siRNA prevent the phenotypic changes observed in podocytes. We will map the binding motifs in Nef that are responsible for its down-stream cellular effects. The proposed project should help us to elucidate essential pathology by which Nef induces altered phenotype in kidney podocytes and its role in the pathogenesis of HIVAN.